Drug conjugate composition

ABSTRACT

The invention provides a liquid composition and a lyophilized composition comprising a therapeutically effective amount of a conjugate comprising an antibody chemically coupled to a maytansinoid. The invention further provides a method for killing a cell in a human comprising administering to the human either of the compositions such that the antibody binds to the surface of the cell and the cytotoxicity of the maytansinoid is activated, whereby the cell is killed.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This patent application is a divisional of U.S. patent application Ser.No. 10/846,129, filed May 14, 2004, which claims the benefit of U.S.Provisional Patent Application No. 60/470,550, filed May 14, 2003.

FIELD OF THE INVENTION

This invention pertains to a conjugate comprising an antibody chemicallycoupled to a maytansinoid, and methods of using same.

BACKGROUND OF THE INVENTION

The treatment of cancer has progressed significantly with thedevelopment of pharmaceuticals that more efficiently target and killcancer cells. To this end, researchers have taken advantage ofcell-surface receptors and antigens selectively expressed by cancercells to develop drugs based on antibodies that bind the tumor-specificor tumor-associated antigens. In this regard, cytotoxic molecules suchas bacteria and plant toxins, radionuclides, and certainchemotherapeutic drugs have been chemically linked to monoclonalantibodies that bind tumor-specific or tumor-associated cell surfaceantigens (see, e.g., International (PCT) Patent Application Nos. WO00/02587, WO 02/060955, and WO 02/092127, U.S. Pat. Nos. 5,475,092,6,340,701, 6,171,586, U.S. Patent Application Publication No.2003/0004210 A1, and Ghetie et al., J. Immunol. Methods, 112, 267-277(1988)). Such compounds are typically referred to as toxin,radionuclide, and drug “conjugates,” respectively. Often they also arereferred to as immunoconjugates, radioimmunoconjugates and immunotoxins.Tumor cell killing occurs upon binding of the drug conjugate to a tumorcell and activation of the cytotoxic activity of the maytansinoid. Theselectivity afforded by drug conjugates minimizes toxicity to normalcells, thereby enhancing tolerability of the drug in the patient.

Despite the tumor selectivity afforded by drug conjugates, the use ofdrug conjugates in a clinical context is limited by a number of factors.In this respect, drug conjugate formulations typically are based on aknown formulation of the antibody from which the drug conjugate ismanufactured, without consideration as to what effect the conjugatedcytotoxic molecule may have on the stability of the antibody. As such,current drug conjugate compositions are less stable than compositionscontaining the tumor-specific antibody alone.

Thus, in view of the above, there remains a need for drug conjugatecompositions containing highly cytotoxic drugs that are more stable thancurrently available drug conjugate compositions. There also remains aneed for methods of using such drug conjugate compositions to treathuman diseases associated with cell proliferation, such as cancer.

The invention provides such a composition and method. These and otheradvantages of the invention, as well as additional inventive features,will be apparent from the description of the invention provided herein.

BRIEF SUMMARY OF THE INVENTION

The invention provides a composition comprising (i) a therapeuticallyeffective amount of a conjugate comprising an antibody chemicallycoupled to a maytansinoid, (ii) a buffering agent, (iii) a tonicifyingamount of sodium chloride, (iv) water, and optionally (v) a surfactant,wherein the composition has a pH of about 5-6. The invention alsoprovides a lyophilized composition comprising (i) a therapeuticallyeffective amount of a conjugate comprising an antibody chemicallycoupled to a maytansinoid, (ii) a buffering agent, (iii) acryoprotectant, (iv) a bulking agent, and optionally (v) a surfactant,wherein the composition has a pH of about 5-6 when reconstituted withwater. The invention further provides a method for killing a cell in ahuman comprising administering to the human either of theabove-described compositions such that the antibody binds to the surfaceof the cell and the cytotoxicity of the maytansinoid is activated,whereby the cell is killed.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a composition comprising (i) a therapeuticallyeffective amount of a conjugate comprising an antibody chemicallycoupled to a maytansinoid, (ii) a buffering agent, (iii) optionally asurfactant, (iv) a tonicifying amount of sodium chloride, and (v) water,wherein the composition has a pH of about 5-6.

The inventive composition contains a conjugate which comprises anantibody chemically coupled to a maytansinoid. The term “antibody,” asused herein, refers to any immunoglobulin, any immunoglobulin fragment,such as Fab, F(ab′)₂, dsFv, sFv, diabodies, and triabodies, orimmunoglobulin chimera, which can bind to an antigen on the surface of acell (e.g., which contains a complementarity determining region (CDR)).Any suitable antibody can be used in the inventive composition. One ofordinary skill in the art will appreciate that the selection of anappropriate antibody will depend upon the cell population. to betargeted. In this regard, the type and number of cell surface molecules(i.e., antigens) that are selectively expressed in a particular cellpopulation (typically and preferably a diseased cell population) willgovern the selection of an appropriate antibody for use in the inventivecomposition. Cell surface expression profiles are known for a widevariety of cell types, including tumor cell types, or, if unknown, canbe determined using routine molecular biology and histochemistrytechniques.

The antibody can be polyclonal or monoclonal, but is most preferably amonoclonal antibody. As used herein, “polyclonal” antibodies refer toheterogeneous populations of antibody, typically contained in the seraof immunized animals. “Monoclonal” antibodies refer to homogenouspopulations of antibody molecules that are specific to a particularantigen. Monoclonal antibodies are typically produced by a single cloneof B lymphocytes (“B cells”). Monoclonal antibodies may be obtainedusing a variety of techniques known to those skilled in the art,including standard hybridoma technology (see, e.g., Köhler and Milstein,Eur. J. Immunol., 5, 511-519 (1976), Harlow and Lane (eds.), Antibodies:A Laboratory Manual, CSH Press (1988), and C. A. Janeway et al. (eds.),Immunobiology, 5^(th) Ed., Garland Publishing, New York, N.Y. (2001)).In brief, the hybridoma method of producing monoclonal antibodiestypically involves injecting any suitable animal, typically andpreferably a mouse, with an antigen (i.e., an “immunogen”). The animalis subsequently sacrificed, and B cells isolated from its spleen arefused with human myeloma cells. A hybrid cell is produced (i.e., a“hybridoma”), which proliferates indefinitely and continuously secreteshigh titers of an antibody with the desired specificity in vitro. Anyappropriate method known in the art can be used to identify hybridomacells that produce an antibody with the desired specificity. Suchmethods include, for example, enzyme-linked immunosorbent assay (ELISA),Western blot analysis, and radioimmunoassay. The population ofhybridomas is screened to isolate individual clones, each of whichsecrete a single antibody species to the antigen. Because each hybridomais a clone derived from fusion with a single B cell, all the antibodymolecules it produces are identical in structure, including theirantigen binding site and isotype. Monoclonal antibodies also may begenerated using other suitable techniques including EBV-hybridomatechnology (see, e.g., Haskard and Archer, J. Immunol. Methods, 74(2),361-67 (1984), and Roder et al., Methods Enzymol., 121, 140-67 (1986)),or bacteriophage vector expression systems (see, e.g., Huse et al.,Science, 246, 1275-81 (1989)). To prepare monoclonal antibody fragments,recombinant methods typically are employed.

The monoclonal antibody can be isolated from or produced in any suitableanimal, but is preferably produced in a mammal, more preferably a mouse,and most preferably a human. Methods for producing an antibody in miceare well known to those skilled in the art and are described herein.With respect to human antibodies, one of ordinary skill in the art willappreciate that polyclonal antibodies can be isolated from the sera ofhuman subjects vaccinated or immunized with an appropriate antigen.Alternatively, human antibodies can be generated by adapting knowntechniques for producing human antibodies in non-human animals such asmice (see, e.g., U.S. Pat. Nos. 5,545,806, 5,569,825, and 5,714,352, andU.S. Patent Application Publication No. 2002/0197266 A1).

While being the ideal choice for therapeutic applications in humans,human antibodies, particularly human monoclonal antibodies, typicallyare more difficult to generate than mouse monoclonal antibodies. Mousemonoclonal antibodies, however, induce a rapid host antibody responsewhen administered to humans, which can reduce the therapeutic ordiagnostic potential of the antibody-drug conjugate. To circumvent thesecomplications, a monoclonal antibody preferably is not recognized as“foreign” by the human immune system. To this end, phage display can beused to generate the antibody. In this regard, phage libraries encodingantigen-binding variable (V) domains of antibodies can be generatedusing standard molecular biology and recombinant DNA techniques (see,e.g., Sambrook et al. (eds.), Molecular Cloning, A Laboratory Manual,3^(rd) Edition, Cold Spring Harbor Laboratory Press, New York (2001)).Phage encoding a variable region with the desired specificity areselected for specific binding to the desired antigen, and a completehuman antibody is reconstituted comprising the selected variable domain.Nucleic acid sequences encoding the reconstituted antibody areintroduced into a suitable cell line, such as a myeloma cell used forhybridoma production, such that human antibodies having thecharacteristics of monoclonal antibodies are secreted by the cell (see,e.g., Janeway et al., supra, Huse et al., supra, and U.S. Pat. No.6,265,150). Alternatively, monoclonal antibodies can be generated frommice that are transgenic for specific human heavy and light chainimmunoglobulin genes. Such methods are known in the art and describedin, for example U.S. Pat. Nos. 5,545,806 and 5,569,825, and Janeway etal., supra. Most preferably the antibody is a humanized antibody. Asused herein, a “humanized” antibody is one in which thecomplementarity-determining regions (CDR) of a mouse monoclonalantibody, which form the antigen binding loops of the antibody, aregrafted onto the framework of a human antibody molecule. Owing to thesimilarity of the frameworks of mouse and human antibodies, it isgenerally accepted in the art that this approach produces a monoclonalantibody that is antigenically identical to a human antibody but bindsthe same antigen as the mouse monoclonal antibody from which the CDRsequences were derived. Methods for generating humanized antibodies arewell known in the art and are described in detail in, for example,Janeway et al., supra, U.S. Pat. Nos. 5,225,539, 5,585,089 and5,693,761, European Patent No. 0239400 B1, and United Kingdom Patent No.2188638. Humanized antibodies can also be generated using the antibodyresurfacing technology described in U.S. Pat. No. 5,639,641 and Pedersenet al., J. Mol. Biol., 235, 959-973 (1994). While the antibody employedin the conjugate of the inventive composition most preferably is ahumanized monoclonal antibody, a human monoclonal antibody or a mousemonoclonal antibody, as described above, are also within the scope ofthe invention.

Antibody fragments that have at least one antigen binding site, and thusrecognize and bind to at least one antigen or receptor present on thesurface of a target cell, also are within the scope of the invention. Inthis respect, proteolytic cleavage of an intact antibody molecule canproduce a variety of antibody fragments that retain the ability torecognize and bind antigens. For example, limited digestion of anantibody molecule with the protease papain typically produces threefragments, two of which are identical and are referred to as the Fabfragments, as they retain the antigen binding activity of the parentantibody molecule. Cleavage of an antibody molecule with the enzymepepsin normally produces two antibody fragments, one of which retainsboth antigen-binding arms of the antibody molecule, and is thus referredto as the F(ab′)₂ fragment. A single-chain variable region fragment(sFv) antibody fragment, which consists of a truncated Fab fragmentcomprising the variable (V) domain of an antibody heavy chain linked toa V domain of a light antibody chain via a synthetic peptide, can begenerated using routine recombinant DNA technology techniques (see,e.g., Janeway et al., sura). Similarly, disulfide-stabilized variableregion fragments (dsFv) can be prepared by recombinant DNA technology(see, e.g., Reiter et al., Protein Engineering, 7, 697-704 (1994)).Antibody fragments of the present invention, however, are not limited tothese exemplary types of antibody fragments. Any suitable antibodyfragment that recognizes and binds to a desired cell surface receptor orantigen can be employed. Antibody-antigen binding can be assayed usingany suitable method known in the art, such as, for example,radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, andcompetitive inhibition assays (see, e.g., Janeway et al., supra, andU.S. Patent Application Publication No. 2002/0197266 A1).

In addition, the antibody can be a chimeric antibody. By “chimeric” ismeant that the antibody comprises at least two immunoglobulins, orfragments thereof, obtained or derived from at least two differentspecies (e.g., two different immunoglobulins, a human immunoglobulinconstant region combined with a murine immunoglobulin variable region).

Any suitable antibody can be used in the inventive composition.Particularly preferred antibodies are humanized monoclonal antibodies,examples of which include huN901, huMy9-6, huB4, huC242, trastuzumab,bivatuzumab, sibrotuzumab, and rituximab (see, e.g., U.S. Pat. No.5,639,641, U.S. Provisional Patent Application No. 60/424,332,International (PCT) Patent Application No. WO 02/16401, Pedersen et al.,J. Mol. Biol., 235, 959-973 (1994), Roguska et al., Proc. Natl. Acad.Sci. USA, 91, 969-73 (1994), Liu et al., Proc. Natl. Acad. Sci. USA, 93,8618-8623 (1996), Nadler et al., J. Immunol., 131, 244-250 (1983),Colomer et al., Cancer Invest., 19, 49-56 (2001), Heider et al., Eur. J.Cancer, 31A, 2385-2391 (1995), Welt et al., J. Clin. Oncol., 12,1193-1203 (1994), Maloney et al., Blood, 90, 2188-2195 (1997), and U.S.Pat. No. 5,665,357). Most preferably, the antibody is the huN901humanized monoclonal antibody or the huMy9-6 humanized monoclonalantibody. Other humanized monoclonal antibodies are known in the art andcan be used in connection with the inventive composition.

In accordance with the invention, the above-described antibody ischemically coupled to any suitable cytotoxic agent, particularly acytotoxic agent that induces cytotoxicity of tumor cells, to form aconjugate as described above. As a result of normal pharmacologicclearance mechanisms, an antibody employed in a drug conjugate contactsand binds to target cells only in limited amounts. Therefore, thecytotoxic agent employed in the conjugate must be highly cytotoxic suchthat sufficient cell killing occurs to elicit a therapeutic effect.Examples of such cytotoxic agents include novel taxanes (see, e.g.,International (PCT) Patent Application Nos. WO 01/38318 andPCT/US03/02675), DNA-alkylating agents (e.g., CC-1065 analogs),anthracyclines, tubulysin analogs, duocarmycin analogs, auristatin E,and cytotoxic agents comprising a reactive polyethylene glycol moiety(see, e.g., Sasse et al., J. Antibiot. (Tokyo), 53, 879-85 (2000),Suzawa et al., Bioorg. Med. Chem., 8, 2175-84 (2000), Ichimura et al.,J. Antibiot. (Tokyo), 44, 1045-53 (1991), Francisco et al., Blood (2003)(electronic publication prior to print publication), U.S. Pat. Nos.5,475,092, 6,340,701, 6,372,738, and 6,436,931, U.S. Patent ApplicationPublication No. 2001/0036923 A1, Pending U.S. patent application Ser.Nos. 10/024,290 and 10/116,053, and International (PCT) PatentApplication No. WO 01/49698). Alternatively and most preferably, theantibody is chemically coupled to a maytansinoid to form the conjugateof the inventive composition.

Maytansinoids were originally isolated from the east African shrubbelonging to the genus Maytenus, but were subsequently also discoveredto be metabolites of soil bacteria, such as Actinosynnema pretiosum(see, e.g., U.S. Pat. No. 3,896,111). Maytansinoids induce cytotoxicitythrough mitotic inhibition. Experimental evidence suggests thatmaytansinoids inhibit mitosis by inhibiting polymerization of themicrotubule protein tubulin, thereby preventing formation ofmicrotubules (see, e.g., U.S. Pat. No. 6,441,163 and Remillard et al.,Science, 189, 1002-1005 (1975)). Maytansinoids have been shown toinhibit tumor cell growth in vitro using cell culture models, and invivo using laboratory animal systems. Moreover, the cytotoxicity ofmaytansinoids is 1,000-fold greater than conventional chemotherapeuticagents, such as, for example, methotrexate, daunorubicin, andvincristine (see, e.g., U.S. Pat. No. 5,208,020). Maytansinoids areknown in the art to include maytansine, maytansinol, C-3 esters ofmaytansinol, and other maytansinol analogues and derivatives (see, e.g.,U.S. Pat. Nos. 5,208,020 and 6,441,163). C-3 esters of maytansinol canbe naturally occurring or synthetically derived. Moreover, bothnaturally occurring and synthetic C-3 maytansinol esters can beclassified as a C-3 ester with simple carboxylic acids, or a C-3 esterwith derivatives of N-methyl-L-alanine, the latter being more cytotoxicthan the former. Synthetic maytansinoid analogues also are known in theart and described in, for example, Kupchan et al., J. Med. Chem., 21,31-37 (1978). Methods for generating maytansinol and analogues andderivatives thereof are described in, for example, U.S. Pat. No.4,151,042.

Suitable maytansinoids for use in the inventive composition can beisolated from natural sources, synthetically produced, orsemi-synthetically produced using methods known in the art. Moreover,the maytansinoid can be modified in any suitable manner, so long assufficient cytotoxicity is preserved in the ultimate conjugate molecule.In this regard, maytansinoids lack suitable functional groups to whichantibodies can be linked. A linking moiety desirably is utilized to linkthe maytansinoid to the antibody to form the conjugate. The linkingmoiety contains a chemical bond that allows for the activation ofmaytansinoid cytotoxicity at a particular site. Suitable chemical bondsare well known in the art and include disulfide bonds, acid labilebonds, photolabile bonds, peptidase labile bonds, thioether bonds formedbetween sulfhydryl and maleimide groups, and esterase labile bonds. Mostpreferably, the linking moiety comprises a disulfide bond or a thioetherbond. In accordance with the invention, the linking moiety preferablycomprises a reactive chemical group. Particularly preferred reactivechemical groups are N-succinimidyl esters and N-sulfosuccinimidylesters. In a preferred embodiment, the reactive chemical group can becovalently bound to the maytansinoid via disulfide bonding between thiolgroups. Thus, a maytansinoid modified as described herein preferablycomprises a thiol group. One of ordinary skill in the art willappreciate that a thiol group contains a sulfur atom bonded to ahydrogen atom and is typically also referred to in the art as asulfhydryl group, which can be denoted as “—SH” or “RSH.”

Particularly preferred maytansinoids comprising a linking moiety thatcontains a reactive chemical group are C-3 esters of maytansinol and itsanalogs where the linking moiety contains a disulfide bond and thechemical reactive group comprises a N-succinimidyl orN-sulfosuccinimidyl ester. Many positions on maytansinoids can serve asthe position to chemically link the linking moiety. For example, the C-3position having a hydroxyl group, the C-14 position modified withhydroxymethyl, the C-15 position modified with hydroxy and the C-20position having a hydroxy group are all useful. The linking moiety mostpreferably is linked to the C-3 position of maytansinol. Mostpreferably, the maytansinoid used in connection with the inventivecomposition isN^(2′)-deacetyl-N^(2′)-(3-mercapto-1-oxopropyl)-maytansine (DM1) orN^(2′)-deacetyl-N^(2′)-(4-mercapto-4-methyl-1-oxopentyl)-maytansine(DM4).

Linking moieties with other chemical bonds also can be used in thecontext of the invention, as can other maytansinoids. Specific examplesof other chemical bonds include acid labile bonds, thioether bonds,photolabile bonds, peptidase labile bonds and esterase labile bonds.Methods for producing maytansinoids with linking moieties are describedin, for example, U.S. Pat. Nos. 5,208,020, 5,416,064, and 6,333,410.

The linking moiety of a maytansinoid typically and preferably is part ofa larger linker molecule that is used to join the antibody to themaytansinoid. Any suitable linker molecule can be used in connectionwith the invention, so long as the linker molecule provides forretention of the cytotoxicity and targeting characteristics of themaytansinoid and the antibody, respectively. The linker molecule joinsthe maytansinoid to the antibody through chemical bonds (as describedabove), such that the maytansinoid and the antibody are chemicallycoupled (e.g., covalently bonded) to each other. Desirably, the linkermolecule chemically couples the maytansinoid to the antibody throughdisulfide bonds or thioether bonds. Most preferably, the antibody ischemically coupled to the maytansinoid via disulfide bonds.

Particularly preferred linker molecules include, for example,N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) (see, e.g., Carlssonet al., Biochem. J., 173, 723-737 (1978)), N-succinimidyl4-(2-pyridyldithio)butanoate (SPDB) (see, e.g., U.S. Pat. No.4,563,304), N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP) (see,e.g., CAS Registry number 341498-08-6), N-succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) (see, e.g.,Yoshitake et al., Eur. J. Biochem., 101, 395-399 (1979)), andN-succinimidyl 4-methyl-4-[2-(5-nitro-pyridyl)-dithio]pentanoate (SMNP)(see, e.g., U.S. Pat. No. 4,563,304) The most preferred linker moleculesfor use in the inventive composition are SPP, SMCC, and SPDB.

The inventive composition comprises a therapeutically effective amountof a conjugate comprising an antibody chemically coupled to amaytansinoid. A “therapeutically effective amount” means an amountsufficient to show a meaningful benefit in an individual, e.g.,promoting at least one aspect of tumor cell cytotoxicity, or treatment,healing, prevention, or amelioration of other relevant medicalcondition(s) associated with a particular cancer. Therapeuticallyeffective amounts may vary depending upon the biological effect desiredin the individual, condition to be treated, and/or the specificcharacteristics of the conjugate, and the individual. Thus, inaccordance with the methods described herein, the attending physician(or other medical professional responsible for administering thecomposition) will typically decide the amount of the composition withwhich to treat each individual patient. The concentration of theconjugate in the inventive composition desirably is about 0.1 mg/mL toabout 5 mg/mL (e.g., about 0.5 mg/mL, about 2 mg/mL, or about 5 mg/mL).In a preferred embodiment, the concentration of the conjugate in theinventive composition is about 1 mg/mL or higher (e.g., about 2 mg/mL orhigher, about 3 mg/mL or higher, or about 4 mg/mL or higher). Mostpreferably, the concentration of the conjugate in the inventivecomposition is about 5 mg/mL. While compositions comprising at least 1mg/mL of the conjugate are particularly preferred, conjugateconcentrations of less than 1 mg/mL (e.g., concentrations of about 0.1mg/mL to about 0.9 mg/mL) also can be stably maintained in the inventivecomposition, and thus are within the scope of the invention.Compositions comprising greater than 1 mg/mL of the conjugate moleculeare advantageous for clinical and commercial use, in that suchconcentrations enable single doses of the composition to be prepared ina more convenient (i.e., smaller) volume for administration.

The inventive composition desirably is formulated to be acceptable forpharmaceutical use, such as, for example, administration to a human hostin need thereof. To this end, the conjugate molecule preferably isformulated into a composition comprising a physiologically acceptablecarrier (e.g., excipient or diluent). Physiologically acceptablecarriers are well known and are readily available, and include bufferingagents, anti-oxidants, bacteriostats, salts, and solutes that render theformulation isotonic with the blood or other bodily fluid of the humanpatient, and aqueous and non-aqueous sterile suspensions that caninclude suspending agents, solubilizers, thickening agents, stabilizers(e.g., surfactants), and preservatives. The choice of carrier will bedetermined, at least in part, by the location of the target tissueand/or cells, and the particular method used to administer thecomposition. Examples of suitable carriers and excipients for use indrug conjugate formulations are disclosed in, for example, International(PCT) Patent Application Nos. WO 00/02587, WO 02/060955, and WO02/092127, and Ghetie et al., J. Immunol. Methods, 112, 267-277 (1988).Most preferably, the inventive composition comprises a buffering agent,a surfactant, a tonicifying amount of sodium chloride, and water.

Any suitable pharmaceutically acceptable buffering agent may be used inconnection with the inventive composition. Examples of particularlypreferred buffering agents include citrate, acetate, succinate,phosphate, and histidine. The inventive composition, however, is notlimited to these exemplary buffering agents. The buffering agent may bepresent in the inventive composition in any suitable concentration, solong as sufficient stability of the composition is achieved under thedesired conditions. In this regard, the concentration of the bufferingagent in the composition preferably is about 2 mM to about 50 mM (e.g.,about 2-10 mM, about 10-20 mM, about 20-30 mM, about 30-40 mM, or about40-50 mM). Most preferably, the concentration of the buffering agent inthe composition is about 5-15 mM (e.g., about 10 mM). The bufferingagent desirably is sodium succinate or sodium acetate, but mostpreferably is sodium citrate. The buffering agent typically is presentin the inventive composition such that the pH is maintained within adesired range. In this respect, the inventive composition preferably hasa pH of about 5-6 (e.g., about 5, 5.5, or 6). It is believed thatcompositions with a higher pH (e.g., about pH 6 or higher) are lessstable than compositions with a lower pH (i.e., about pH 6 or less).Thus, the inventive composition most preferably has a pH of about 5.5.

In addition to the buffering agent discussed above, the inventivecomposition also optionally contains a surfactant. Any suitablesurfactant can be used in connection with the invention. Suitablesurfactants are well known to those skilled in the art. In accordancewith the inventive composition, the surfactant desirably is apolysorbate, and preferably is polysorbate 20 or polysorbate 80. Mostpreferably, the surfactant is polysorbate 20. The surfactant may bepresent in the inventive composition in any suitable concentration, solong as sufficient stability of the composition is achieved under thedesired conditions. In this regard, the concentration of the surfactantin the composition preferably is about 0.002% to about 0.1% wt./vol.(e.g., about 0.002-0.01%, about 0.005-0.02%, or about 0.01-0.1%wt./vol.) of the total volume of the composition. Most preferably, theconcentration of the surfactant in the composition is about 0.005-0.02%wt./vol. (e.g., about 0.01% wt./vol.) of the total volume of thecomposition. Although compositions formulated with surfactants arepreferred, compositions formulated without surfactants are also withinthe scope of the invention.

As an additional stabilizing agent, sodium chloride also is added to theinventive composition. In this regard, the inventive compositioncomprises a suitable amount, preferably a tonicifying amount, of sodiumchloride (NaCl). By the phrase a “tonicifying amount of sodiumchloride,” it is meant that the concentration of NaCl in the compositionis such that the tonicity of the composition is the same as the tonicityof human blood (i.e., isotonic). In this regard, the NaCl can be presentin the inventive composition in any suitable concentration, so long assufficient tonicity and stability is achieved in the inventivecomposition. Desirably, the concentration of sodium chloride in thecomposition is about 50 mM to about 500 mM (e.g., about 50-100 mM, about100-150 mM, about 150-250 mM, about 250-350 mM, or about 350-450 mM).While higher concentrations of sodium chloride (e.g., about 150 mM ormore) may render the inventive composition hypertonic rather thanisotonic, dilution of such compositions with any suitable isotonicsolvent such as, preferably, dextrose 5% in water (“D5W”) or normalsaline (“NS”) prior to human administration would render suchcompositions only slightly hypertonic and suitable for use in theinvention. Preferably, the concentration of sodium chloride in thecomposition is about 100 mM to about 200 mM (e.g., about 100-140 mM,about 130-170 mM, or about 160-200 mM). Most preferably, theconcentration of sodium chloride in the composition is about 110-150 mM(e.g., about 110 mM-130 mM, or about 120 mM).

In a particularly preferred embodiment of the invention, the compositioncomprises (i) about 5 mg/mL of a conjugate comprising huN901 chemicallycoupled to DM1, (ii) about 10 mM sodium citrate buffer, (iii) about0.01% polysorbate 20, (iv) about 120 mM sodium chloride, and (v) water(preferably water suitable for injection (WFI)), wherein the pH of thecomposition is about 5.5. In another preferred embodiment, thecomposition comprises (i) about 1 mg/mL or more (e.g., about 1 mg/mL,about 2 mg/mL, 3 mg/mL, about 5 mg/mL, or ranges therebetween) of aconjugate comprising huMy9-6 chemically coupled to DM1, (ii) about 10 mMsodium citrate buffer, (iii) optionally about 0.01% polysorbate 20, (iv)about 135 mM sodium chloride, and (v) water, wherein the pH of thecomposition is about 5.5. In yet another preferred embodiment, thecomposition comprises (i) about 1 mg/mL or more (e.g., about 1 mg/mL,about 2 mg/mL, 3 mg/mL, about 5 mg/mL, or ranges therebetween) of aconjugate comprising huMy9-6 chemically coupled to DM4, (ii) about 10 mMsodium citrate buffer, (iii) optionally about 0.01% polysorbate 20, (iv)about 135 mM sodium chloride, and (v) water, wherein the pH of thecomposition is about 5.5. In an additional preferred embodiment, thecomposition comprises (i) about 1 mg/mL or more (e.g., about 1 mg/mL,about 2 mg/mL, 3 mg/mL, about 5 mg/mL, or ranges therebetween) of aconjugate comprising huN901 chemically coupled to DM1 via an SMCClinker, (ii) about 10 mM sodium citrate buffer, (iii) optionally about0.01% polysorbate 20, (iv) about 130 mM sodium chloride, and (v) water,wherein the pH of the composition is about 5.5.

Compositions containing antibodies (or proteins in general) are renderedunstable by oxidation. Thus, in another embodiment of the invention, thecomposition further comprises an antioxidant. Any suitable antioxidantcan be used in the inventive composition. Suitable antioxidants areknown in the art and include, for example, superoxide dismutase,glutathione peroxidase, tocotrienols, polyphenols, zinc, manganese,selenium, vitamin C, vitamin E, beta carotene, cysteine, and methionine.The antioxidant used in connection with the inventive composition mostpreferably is methionine. The antioxidant can be present in thecomposition in any suitable concentration. Desirably, the concentrationof the antioxidant in the composition is about 100 μM to about 100 mM(e.g., about 0.25-1 mM, about 0.5-2 mM, about 5-15 mM, about 20-70 mM,or about 60-90 mM). Most preferably, the concentration of theantioxidant in the composition is about 5-15 mM (e.g., about 10 mM).

In addition to antioxidants, the inventive composition can further bestabilized by the addition of sucrose. The use of sucrose to stabilizeantibody formulations is known to those of skill in the art. Anysuitable amount of sucrose can be used in the inventive composition, butthe concentration of sucrose in the composition desirably is about 0.1%to about 10% wt./vol. (e.g., about 0.1-1%, about 2-5%, or about 7-10%wt./vol.) of the total volume of the composition. Most preferably, theconcentration of sucrose in the composition is about 4-6% wt./vol.(e.g., about 5% wt./vol.) of the total volume of the composition.

The invention further provides a packaged composition comprising asealed container having the inventive composition dispersed therein, andan inert gas overlay. The packaged composition can be overlaid with anysuitable inert gas, so long as the inventive composition is stablymaintained within the packaged composition. The inert gas preferably isnitrogen or argon. The packaged composition can be presented inunit-dose or multi-dose sealed containers, such as ampules or vials.

In addition to the water-containing composition described herein (alsoreferred to herein as a “liquid” or “aqueous” composition), theinvention also provides a lyophilized composition comprising (i) atherapeutically effective amount of a conjugate comprising an antibodychemically coupled to a maytansinoid, (ii) a buffering agent, (iii) asurfactant, (iv) a cryoprotectant, and (v) a bulking agent, wherein thecomposition has a pH of about 5-6 when reconstituted with water. By“lyophilized” is meant that the composition has been freeze-dried undera vacuum. Lyophilization typically is accomplished by freezing aparticular formulation such that the solutes are separated from thesolvent(s). The solvent is then removed by sublimation (i.e., primarydrying) and next by desorption (i.e., secondary drying). Descriptions ofthe conjugate (i.e., the antibody chemically coupled to themaytansinoid), buffering agent, surfactant, and components thereof, setforth above in connection with other embodiments of the invention alsoare applicable to those same aspects of the aforesaid lyophilizedcomposition. Prior to reconstitution of the lyophilized composition, therelative amounts of each component comprising the inventive lyophilizedcomposition can be described in terms of mg of excipient (e.g., buffer,surfactant, bulking agent, cryoprotectant) per mg of conjugate.

While any suitable buffering agent described herein can be used inconnection with the inventive lyophilized composition, the inventivelyophilized composition preferably comprises a sodium succinate buffer.The buffering agent can be present in the inventive lyophilizedcomposition in any suitable amount. In particular, the lyophilizedcomposition desirably comprises about 0.1 mg to about 2 mg of thebuffering agent per mg of the conjugate (e.g., about 0.1 mg to about 0.5mg buffering agent per mg of the conjugate, about 0.5 mg to about 1 mgbuffering agent per mg of the conjugate, or about 1 mg about 2 mgbuffering agent per mg of the conjugate). Most preferably, thelyophilized composition comprises about 0.3 mg sodium succinate bufferper mg of the conjugate.

While any suitable surfactant described herein can be used in connectionwith the inventive lyophilized composition, the surfactant desirably isa polysorbate, and preferably is polysorbate 20 or polysorbate 80. Mostpreferably, the surfactant is polysorbate 20. The surfactant may bepresent in the inventive lyophilized composition in any suitable amount,so long as sufficient stability of the lyophilized composition isachieved under the desired conditions. In this regard, the lyophilizedcomposition desirably comprises about 0.005 mg to about 0.1 mg of thesurfactant per mg of the conjugate (e.g., about 0.005 mg to about 0.01mg surfactant per mg of the conjugate, about 0.01 mg to about 0.05 mgsurfactant per mg of the conjugate, or about 0.05 mg to about 0.1 mgsurfactant per mg of the conjugate). When the surfactant is polysorbate20, the lyophilized composition preferably comprises about 0.02 mgpolysorbate 20 per mg of the conjugate.

In order to prevent degradation of the active ingredients of thecomposition during freezing and drying, the inventive lyophilizedcomposition further comprises a cryoprotectant, preferably an amorphouscryoprotectant. The term “cryoprotectant,” as used herein, refers to anexcipient that protects unstable molecules during freezing. Suitablecryoprotectants for use in the inventive composition are known to thoseskilled in the art, and include, for example, glycerol, dimethylsulfoxide (DMSO), polyethylene glycol (PEG), dextran, glucose,trehalose, and sucrose. Most preferably, the cryoprotectant is sucrose.The cryoprotectant may be present in the inventive lyophilizedcomposition in any suitable amount. The lyophilized compositiondesirably comprises about 0.5 mg to about 5 mg (e.g., about 0.5 mg toabout 2 mg) of the cryoprotectant per mg of the conjugate (e.g., about0.8 mg cryoprotectant per mg of the conjugate, about 2 mg cryoprotectantper mg of the conjugate, or about 4 mg cryoprotectant per mg of theconjugate). When the cryoprotectant is sucrose, the lyophilizedcomposition preferably comprises about 0.5 mg to about 2 mg (e.g., about1 mg) sucrose per mg of the conjugate.

The inventive lyophilized composition can further contain a bulkingagent, preferably a crystallizable bulking agent. Bulking agentstypically are used in the art to provide structure and weight to the“cake” produced as a result of lyophilization. Any suitable bulkingagent known in the art may be used in connection with the inventivelyophilized composition. Suitable bulking agents include, for example,mannitol, dextran, and glycine. The bulking agent used in the inventivecomposition most preferably is glycine. The lyophilized composition cancontain any suitable amount of the bulking agent, but preferably thelyophilized composition comprises about 2 mg to about 20 mg of thebulking agent per mg of the conjugate (e.g., about 2 mg to about 10 mgbulking agent per mg of the conjugate, about 5 mg to about 10 mg bulkingagent per mg of the conjugate, about 10 mg to about 15 mg bulking agentper mg of the conjugate, or about 15 mg to about 20 mg bulking agent permg of the conjugate). When the bulking agent is glycine, the lyophilizedcomposition preferably comprises about 3.8 mg glycine per mg of theconjugate.

Thus, in accordance with the invention, the contents of a lyophilizedcomposition that is to be reconstituted to contain 5 mg/mL of conjugate(e.g., preferably a conjugate comprising huN901 chemically coupled toDM1) preferably comprises (i) about 0.3 mg sodium succinate buffer permg of the conjugate, (ii) about 0.02 mg polysorbate 20 per mg of theconjugate, (iii) about 1 mg sucrose per mg of the conjugate, and (iv)about 3.8 mg glycine per mg of the conjugate. Once reconstituted withwater, such a lyophilized composition preferably has a pH of about 5.5.Moreover, when the lyophilized composition is reconstituted with water,the descriptions of the relative concentrations of the conjugate, thebuffering agent, and the surfactant set forth above in connection withthe inventive liquid composition also are applicable to the aforesaidlyophilized composition.

Lyophilization methods are well known in the art and are described in,for example, Wang, W., Int. J. Pharm., 203, 1-60 (2000). For example,the inventive lyophilized composition can be produced using alyophilization cycle comprising the following steps: (1) pre-cooling ata shelf temperature of 4° C. and ambient chamber pressure for 2.5 hours,(2) freezing at a shelf temperature of −50° C. and ambient chamberpressure for 14 hours, (3) glycine recrystallization at a shelftemperature of −20° C. and ambient chamber pressure for 6 hours, (4)re-freezing at a shelf temperature of −50° C. and ambient chamberpressure for 16 hours, (5) primary drying at a shelf temperature of −13°C. and 100 mTorr of pressure for 24 hours, (6) secondary drying at ashelf temperature of 24° C. and 100 mTorr of pressure for 10 hours, and(7) stopper phase at a shelf temperature of 24° C. and ambient chamberpressure. The inventive lyophilized composition, however, is not limitedto compositions produced by the above-described method. Indeed, anysuitable lyophilization method can be used to produce the inventivelyophilized composition, and it will be apparent to those skilled in theart that the chosen lyophilization parameters (e.g., drying times) willvary depending on a variety of factors, including the volume of thesolution to be lyophilized.

In addition to the preferred embodiments described herein, the inventivecomposition (whether in liquid or lyophilized form) can compriseadditional therapeutic or biologically active agents. For example,therapeutic factors useful in the treatment of a particular indication(e.g., cancer) can be present. Factors that control inflammation, suchas ibuprofen or steroids, can be part of the composition to reduceswelling and inflammation associated with in vivo administration of thecomposition and physiological distress. Immune enhancers can be includedin the composition to up regulate the body's natural defenses againstdisease. Vitamins and minerals, antioxidants, and micronutrients can beco-administered with the composition. Antibiotics, i.e., microbicidesand fungicides, can be present to reduce the risk of infectionpertaining to the procedures associated with administration of thecomposition and other disorders.

The invention further provides a method for killing a cell in a humancomprising administering to the human a composition comprising (i) atherapeutically effective amount of a conjugate comprising an antibodychemically coupled to a maytansinoid, (ii) a buffering agent, (iii) asurfactant, (iv) a tonicifying amount of sodium chloride, and (v) water,wherein the composition has a pH of about 5-6, such that the antibodybinds to the surface of the cell and the cytotoxicity of themaytansinoid is activated, whereby the cell is killed. Descriptions ofthe conjugate (i.e., the antibody chemically coupled to themaytansinoid), excipients (e.g., buffering agent, surfactant, sodiumchloride, etc.), and components thereof, set forth above in connectionwith other embodiments of the invention also are applicable to thosesame aspects of the aforesaid inventive method.

The inventive method involves administering the inventive composition toa human. Ideally, the inventive method is used to target and kill cellsaffected by a disease, particularly a disease associated with elevatedlevels of cellular proliferation, such as cancer. Thus, in this regard,the inventive method preferably is used to kill tumor cells in a human,thereby resulting in the prevention, amelioration, and/or cure of thecancer.

While any suitable means of administering the composition to a human canbe used within the context of the invention, typically and preferablythe inventive composition is administered to a human via injection, andmost preferably via infusion. By the term “injection,” it is meant thatthe composition is forcefully introduced into a target tissue of thehuman. By the term “infusion,” it is meant that the composition isintroduced into a tissue, typically and preferably a vein, of the human.The composition can be administered to the human by any suitable route,but preferably is administered to the human intravenously orintraperitoneally. When the inventive method is employed to kill tumorcells, however, intratumoral administration of the inventive compositionis particularly preferred. When the inventive composition isadministered by injecting, any suitable injection device can be used toadminister the composition directly to a tumor. For example, the commonmedical syringe can be used to directly inject the composition into asubcutaneous tumor. Alternatively, the composition can be topicallyapplied to the tumor by bathing the tumor in the inventive liquidcomposition. Likewise, the tumor can be perfused with the inventivecomposition over a period of time using any suitable delivery device,e.g., a catheter. While less preferred, other routes of administrationcan be used to deliver the composition to the human. Indeed, althoughmore than one route can be used to administer the inventive composition,a particular route can provide a more immediate and more effectivereaction than another route. For example, while not particularlypreferred, the inventive composition can be applied or instilled intobody cavities, absorbed through the skin, inhaled, or administeredparenterally via, for instance, intramuscular or intraarterialadministration. Preferably, the inventive composition parenterallyadministered to a human is specifically targeted to particular cells,e.g., cancer cells.

As described herein, the conjugate comprises an antibody, which ispreferably a humanized monoclonal antibody, such as huN901, huMy9-6,huB4, or huC242. Other suitable antibodies include, for example,trastuzumab, bivatuzumab, sibrotuzumab, and rituximab. When compositionscomprising such conjugates are employed in the inventive method, theantibody targets the conjugate to a desired cell (e.g., a tumor cell)through interactions with antigens (e.g., tumor-specific antigens)expressed at the surface of the cell (e.g., tumor cell). Tumor-specificantigens have been extensively described in the prior art for a varietyof tumors, including, for example, epithelial cancers (e.g., MUC1), andbreast and ovarian cancer (e.g., HER2/neu), (see, e.g., Bartnes,Tidsskr. Nor. Laegeforen., 121, 2941-5 (2001), and von Mensdorff-Pouillyet al., Int. J. Biol. Markers, 15, 343-356 (2000)).

In a preferred embodiment of the invention, the antibody (e.g., huMy9-6)binds to the CD33 antigen, which is expressed, for example, by acutemyeloid leukemia cells. In another preferred embodiment, the antibody(e.g., huB4) binds to the CD 19 antigen, which is expressed, forexample, by human B-cell lymphoma cells. Alternatively, the antibody(e.g., huC242) binds to the CanAg antigen, which is expressed by anumber of cancer cell types, including, for example, colorectal,pancreatic, gastric, and other gastrointestinal cancers, and themajority of non-small-cell lung cancers. Most preferably, the antibody(e.g., huN901) binds to the NCAM/CD56 antigen, which is expressed, forexample, by small cell lung carcinoma (SCLC) cells, and by other cancersof neuroendocrine origin. Other preferred antigens to which the antibodycan bind include the GD₃ antigen, PSMA, the alpha-folate receptor,Her2/neu, CD44v6, the fetoacinar pancreatic (FAP) antigen, the Cripto-1antigen, the CA6 antigen, CD20, CA 55.1, MN/CA IX, and chondroitinsulfate proteoglycan (see, e.g., Chang et al., Cancer Res., 59, 3192-98(1999), Miotti et al., Int. J. Cancer, 39, 297-303, (1987), Colomer etal., supra, Heider et al., supra, Welt et al., supra, LePage et al.,American Assn. For Cancer Research (AACR), 2003 Anuual Meeting, PosterAbstact No. 749, Kearse et al., Int. J. Cancer, 88, 866-72 (2000),Maloney et al., supra, Opavsky et al., Genomics, 33, 480-87 (1996), Behmet al., Blood, 87, 1134-39 (1996), and U.S. Pat. No. 5,665,357). Uponbinding of the conjugate to a target (i.e., tumor) cell via any of thetumor specific antigens or receptors described herein, the cytotoxicityof the maytansinoid is activated. Examples of mechanisms by whichmaytansinoid cytotoxicity can be activated include release of the freemaytansinoid inside the cell via cleavage of the disulfide linkagebetween the antibody and the maytansinoid, antibody degradation withinthe cell, and activation of maytansinoid cytotoxicity at the cellsurface. The inventive method, however, is not limited to theseexemplary modes of maytansinoid activation. Indeed, any mechanism thatactivates the cytotoxicity of the maytansinoid is within the scope ofthe inventive method.

For the purposes of human administration, the inventive liquidcomposition described herein may be administered (e.g., infused)directly to a human, or diluted with a suitable diluent immediatelyprior to administration. Suitable diluents are known in the art andinclude D5W and normal saline (NS). Dilutions of 1:1, 1:2, 1:3, or more(e.g., 1:5, 1:10, or even 1:50) with suitable diluents are possible.Dilution of the inventive composition desirably does not reduce theconcentration of the conjugate molecule in the composition below about0.1 mg/mL. Upon diluting the inventive liquid composition, thepreviously described concentrations of each of the components (e.g.,buffering agent, surfactant, and sodium chloride) of the composition arecorrespondingly reduced.

When the inventive lyophilized composition described herein isadministered to a human, the composition must be first reconstituted byadding a sterile liquid excipient, for example, water suitable forinjection, D5W, or NS, immediately prior to use. Thus, the inventionfurther provides a method for killing a cell in a human comprising (a)providing the lyophilized composition as described herein, (b) addingwater to the lyophilized composition to provide a reconstitutedcomposition, and (c) administering the reconstituted composition to thehuman such that the antibody binds to the surface of the cell and themaytansinoid is internalized by the cell, whereby the cell is killed.Descriptions of the lyophilized composition, administration routes,tumor specific antigens, and components thereof, set forth above inconnection with other embodiments of the invention also are applicableto those same aspects of the aforesaid inventive method. Moreover, asdiscussed herein, after the inventive lyophilized composition isreconstituted with water, the descriptions of the relativeconcentrations of the conjugate and excipients (e.g., buffering agent,surfactant, cryoprotectant, and bulking agent) described above inconnection with the inventive liquid composition also are applicable tothose same aspects of the aforesaid inventive method.

As discussed herein, the inventive method, whether employing a liquidcomposition or a lyophilized composition, preferably is used inconnection with treating cancer. The inventive method can be used totreat cancer of any type, including, for example, cancer of the lung,breast, colon, prostate, kidney, pancreas, ovary, blood, and lymphaticorgans. While less preferred, the inventive composition may be used totreat other diseases associated with cellular proliferation includingautoimmune diseases (e.g., systemic lupus, rheumatoid arthritis, andmultiple sclerosis), graft rejections (e.g., renal transplant rejection,liver transplant rejection, lung transplant rejection, cardiactransplant rejection, and bone marrow transplant rejection), graftversus host disease, viral infections (e.g., CMV infection, HIVinfection, AIDS, etc,), and parasitic infections (e.g., giardiasis,amoebiasis, schistosomiasis), and others.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

EXAMPLE 1

This example demonstrates the production of a composition comprising aconjugate comprising an antibody chemically coupled to a maytansinoid,buffering agent, surfactant, tonicifying amount of sodium chloride, andwater.

The generation of a conjugate comprising the huN901 monoclonal antibodychemically coupled to the maytansinoid DM1 via disulfide bonds(“huN901:DM1”) has been previously described (see, e.g., U.S. Pat. No.6,441,163). Formulations containing either 1 mg/mL or 5 mg/mL of thehuN901:DM1 conjugate in the presence of each of the above-describedexcipients individually were prepared. The stability of each of theformulations was assessed using the following assays: visual inspectionto detect particulates, a chromatographic method to measure freedrug-related species, and HPLC size exclusion chromatography (SEC-HPLC)to detect high and low molecular weight conjugate-related species. Withrespect to visual inspection, the presence of particulates areindicative of instability and, therefore, are considered undesirable,while a clear solution is indicative of a stable formulation. Thechromatographic assay was used to measure the amount of free drug at 4°C. and 25° C. The results of each of these assays suggested that aformulation containing about 5 mg/mL huN901:DM1 conjugate, about 10 mMsodium citrate, about 0.01% polysorbate and sodium chloride at pH 5.5(i.e., the inventive liquid composition) would have superior stability.

To confirm the stability of the above-described formulation with respectto dimer formation, which also is an indicator of instability, thehuN901:DM1 conjugate was concentrated and formulated in either phosphatebuffered saline (PBS), pH 6.5 (Formulations 1A-1C), or 10 mM sodiumcitrate, 0.01% polysorbate 20, 60 mM NaCl, pH 5.5 (Formulation 1D).After 6 months at 4° C. or 25° C. the samples were assayed by SEC-HPLC.The results of this analysis are set forth in Table 1. TABLE 1 %Conjugate Conjugate Dimer after 6 Concentration Buffering months atFormulation (mg/mL) Agent Surfactant NaCl 4° C. 25° C. 1A 5.0 PBS, pHNone None 5.5 9.3 (comparative) 6.5 1B 3.8 PBS, pH None None 5.5 8.4(comparative) 6.5 1C 1.2 PBS, pH None None 4.9 6.0 (comparative) 6.5 1D5.0 Sodium 0.01% 60 mM 5.1 6.0 (invention) citrate, pH polysorbate 205.5

These results demonstrate that compositions of the invention (asrepresented by Formulation 1D) protected against formation of conjugatedimer. Thus, the combined results of the visual inspection assay, thechromatographic assay, and the SEC-HPLC assay indicate that theinventive composition was the most stable of the tested formulations.

EXAMPLE 2

This example demonstrates the production of a composition comprising aconjugate comprising an antibody chemically coupled to a maytansinoid,buffering agent, surfactant or sucrose, tonicifying amount of sodiumchloride, and water

A conjugate comprising the huN901 monoclonal antibody chemically coupledto the maytansinoid DM1 via an N-succinimidyl4-(2-pyridyldithio)pentanoate (SPP) linker (“huN901-SPP-DM1”) wasprepared using methods described herein and known in the art (see, e.g.,U.S. Pat. No. 6,441,163). The huN901-SPP-DM1 conjugate was formulated ineither (a) PBS, pH 6.5 (Formulations 2A and 2B) or (b) 10 mM sodiumcitrate, 0.01% polysorbate 20, 135 mM NaCl, pH 5.5 (Formulation 2C) atvarying concentrations. Samples of each of the formulations wereincubated at 4° C. and 25° C. for 6 months, after which the formulationswere tested for the presence of free drug and conjugate dimers bychromatographic assays. The results of these analyses are set forth inTable 2. TABLE 2 Conjugate % Dimer % Free Drug Concentration BufferingTime 6 months Time 6 months Formulation (mg/mL) Agent Surfactant Zero 4°C. 25° C. Zero 4° C. 25° C. 2A 1.0 PBS, pH None 4.8 5.8 6.1 1.1 1.6 4.6(Comparative) 6.5 2B 5.0 PBS, pH None 5.2 8.5 10.1 1.1 1.6 4.9(Comparative) 6.5 2C 5.0 Sodium 0.01% 4.4 5.5 6.4 0.4 1.2 2.9(Invention) citrate, pH polysorbate 5.5

In addition to the results recited in Table 2, the formulations werevisually inspected for particulates. The inventive formulation(Formulation 2C) was visually clear after 6 months' storage at 4° C.,while particulates and sediments were observed in the comparativeformulations (as represented by formulations 2A and 2B).

These results demonstrate the enhanced stability of compositions of theinvention.

EXAMPLE 3

This example demonstrates the production of a composition comprising aconjugate comprising an antibody chemically coupled to a maytansinoid,buffering agent, surfactant, tonicifying amount of sodium chloride, anantioxidant, and water.

A conjugate comprising the huN901 monoclonal antibody chemically coupledto the maytansinoid DM1 via an N-succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker(“huN901-SMCC-DM1”) was prepared using methods described herein andknown in the art (see, e.g., U.S. Pat. No. 6,441,163). 1 mg/mLhuN901-SMCC-DM1 conjugate was formulated in either (a) phosphatebuffered saline (PBS), pH 6.5 (Formulation 3A), (b) 10 mM sodiumcitrate, 0.01% polysorbate 20, 130 mM NaCl, pH 5.5 (Formulation 3B), or(c) 10 mM sodium citrate, 0.01% polysorbate 20, 130 mM NaCl, 10 mMmethionine, pH 5.5 (Formulation 3C). After a 3.5-month incubation at 25°C. and 37° C., samples of each of the formulations were tested for thepresence of conjugate dimers by a chromatographic assay. The results ofthis analysis are set forth in Table 3. TABLE 3 % Dimer Buffering Time3.5-months Formulation Agent Surfactant Antioxidant Zero 25° C. 37° C.3A PBS, pH None None 4.0 5.8 11.2 (comparative) 6.5 3B Sodium 0.01% None4.0 4.7 6.3 (invention) citrate, pH 5.5 polysorbate 20 3C Sodium 0.01%10 mM 4.0 4.4 4.8 (invention) citrate, pH polysorbate methionine 5.5 20

These results demonstrate that compositions of the invention (asrepresented by Formulations 3B and 3C) provide enhanced stability.

EXAMPLE 4

This example demonstrates the production of a composition comprising aconjugate comprising the monoclonal antibody huMy9-6 chemically coupledto the maytansinoid DM1, buffering agent, tonicifying amount of sodiumchloride, and water, with or without a surfactant and sucrose.

A conjugate comprising the huMy9-6 monoclonal antibody chemicallycoupled to the maytansinoid DM1 via an N-succinimidyl4-(2-pyridyldithio)pentanoate (SPP) linker (“huMy9-6-SPP-DM1”) wasprepared using methods described herein and known in the art (see, e.g.,U.S. Pat. No. 6,441,163). 1 mg/mL of the huMy9-6-SPP-DM1 conjugate wasformulated in either (a) phosphate buffered saline (PBS), pH 6.5(Formulation 4A), (b) 10 mM sodium citrate, 135 mM NaCl, pH 5.5(Formulation 4B), (c) 10 mM sodium citrate, 0.01% polysorbate 20, 135 mMNaCl, pH 5.5 (Formulation 4C), or (d) 10 mM sodium citrate, 5% sucrose,60 mM NaCl, pH 5.5 (Formulation 4D). After a three-month incubation at4° C. or 25° C., samples of each of the formulations were assayed bySEC-HPLC to measure high molecular weight (HMW) species, and by achromatographic assay to measure free drug species. The results of thisanalysis are set forth in Table 4. TABLE 4 % Free Drug % HMW SpeciesBuffering NaCl Time 3 months Time 3 months Formulation Agent (mM)Surfactant Stabilizer Zero 4° C. 25° C. Zero 4° C. 25° C. 4A PBS, pHNone None None 0.2 1.3 3.2 0.5 1.4 2.0 (comparative) 6.5 4B Sodium 135None None 0.1 1.0 1.8 0.4 0.7 1.4 (invention) citrate, pH 5.5 4C Sodium135 0.01% None 0.1 1.0 1.8 0.5 0.8 1.8 (invention) citrate, pHpolysorbate 5.5 20 4D Sodium 60 None 5% sucrose 0.1 1.1 1.9 0.4 0.5 0.8(invention) citrate, pH 5.5

In addition, samples of Formulations 4A and 4B were incubated at 4° C.and 25° C. for three months, and then tested for conjugate dimerformation as discussed above. The results of this analysis are set forthin Table 5. TABLE 5 % Dimer 3 months Formulation Buffering Agent TimeZero 4° C. 25° C. 4A PBS, pH 6.5 6.3 10.8 12.3 (comparative) 4B Sodiumcitrate, 5.7 7.0 7.7 (invention) pH5.5

These results demonstrate that compositions of the invention (asrepresented by Formulations 4B, 4C, and 4D) provide enhanced stability,and that sucrose adds additional stabilizing benefits.

EXAMPLE 5

This example demonstrates the production of a composition comprising aconjugate comprising the monoclonal antibody huMy9-6 chemically coupledto the maytansinoid DM4, buffering agent, tonicifying amount of sodiumchloride, and water, with or without a surfactant.

A conjugate comprising the huMy9-6 monoclonal antibody chemicallycoupled to the maytansinoid DM4 via an N-succinimidyl4-(2-pyridyldithio)butanoate (SPDB) linker (“huMy9-6-SPDB-DM4”) wasprepared using methods described herein and known in the art (see, e.g.,U.S. Pat. No. 6,441,163). 1 mg/mL of the huMy9-6-SPDB-DM4 conjugate wasformulated in either (a) phosphate buffered saline (PBS), pH 6.5, (b) 10mM sodium citrate, 135 mM NaCl, pH 5.5, or (c) 10 mM sodium citrate,0.01% polysorbate 20, 135 mM NaCl, pH 5.5. To confirm the stability ofthe above-described formulations, samples of each of the formulationswere tested for the presence of particles using a HIAC particle counterafter a six-month incubation at −80° C. Samples of each of theformulations also were tested for the presence of free drug species asdescribed above after a six-month incubation at 4° C. or 25° C. Theresults of these analyses are set forth in Table 6. TABLE 6 >5 μmParticles % Free after 6 Drug after Buffering NaCl months at 6 monthsFormulation Agent Surfactant mM −80° C. 4° C. 25° C. 5A PBS, None None21218 1.6 5.0 (com- pH 6.5 parative) 5B Sodium None 135 8778 1.2 2.5(invention) citrate, pH 5.5 5C Sodium 0.01% 135 776 1.1 2.8 (invention)citrate, pH poly- 5.5 sorbate 20

These results demonstrate that the compositions of the invention (asrepresented by Formulations 5B and 5C) protected against the formationof free drug species and particulates, and that the presence ofpolysorbate added additional stability protection against particleformation.

EXAMPLE 6

This example demonstrates the production of a lyophilized compositioncomprising a conjugate comprising the monoclonal antibody huN901chemically coupled to the maytansinoid DM1.

The generation of a conjugate comprising the huN901 human monoclonalantibody chemically coupled to the maytansinoid DM1 via disulfide bonds(“huN901:DM1”) has been previously described (see, e.g., U.S. Pat. No.6,441,163). Four formulations, designated Formulations 6A-6D, wereprepared. Each formulation contained (a) 1 mg/mL huN901-DM1, (b) either10 mM sodium citrate or 10 mM-sodium succinate, (c) 0.5% wt./vol.sucrose, (d) 250 mM glycine, and (e) water, with or without 0.01%wt./vol. polysorbate 20, at a pH of 5.5, as set forth in Table 7. TABLE7 Cryo- Bulking Formulation Buffering Agent Surfactant protectant Agent6A sodium citrate none sucrose glycine (comparative) 6B sodium citratepolysorbate 20 sucrose glycine (comparative) 6C sodium succinate nonesucrose glycine (comparative) 6D sodium succinate polysorbate 20 sucroseglycine (invention)

1 mL samples of each of the Formulations 6A-6D were lyophilized in 1 mLvials according to the lyophilization scheme recited in Table 8. TABLE 8Shelf Temp Chamber Pressure Step Duration Lyophilization Step (° C.)(mTorr) (hours) Pre-cool 4 Ambient 2.5 Freeze −50 Ambient 14 Glycine −20Ambient 6 recrystallization Re-freeze −50 Ambient 16 Primary dry −13 10024 Secondary dry 24 100 10 Stopper 24 Ambient —

After lyophilization, samples of each of Formulations 6A-6D displayedsolid, uniform white cakes, and samples of all formulations resuspendedrapidly (i.e., less than 20 seconds for complete dissolution) whenreconstituted in distilled water. The reconstituted samples wereanalyzed for visual appearance and high molecular weight species by HPLCsize exclusion chromatography (SEC-HPLC). The presence of particulatesand/or a high molecular weight species are indicative of instabilityand, therefore, are considered undesirable, while a clear solution isindicative of a stable formulation. The results of this analysis are setforth in Table 9. TABLE 9 Post-reconstitution High Molecular FormulationAppearance Weight (%) 6A Opalescent, particles 0.3 (comparative) 6BParticles 0 (comparative) 6C Clear, no particulates 0.39 (comparative)6D Clear, no particulates 0.05 (invention)

Based on these results, the lyophilized composition of the invention(i.e., Formulation 6D) was the only composition that effectivelyprevented the formation of particulates and high molecular weightspecies upon lyophilization. The inventive lyophilized composition wasthe most stable of the tested formulations.

EXAMPLE 7

This example demonstrates the stability of a lyophilized compositioncomprising a conjugate comprising the monoclonal antibody huN901chemically coupled to the maytansinoid DM1.

A conjugate comprising the huN901 monoclonal antibody chemically coupledto the maytansinoid DM1 via an N-succinimidyl4-(2-pyridyldithio)pentanoate (SPP) linker (“huN901-SPP-DM1”) wasprepared as described herein. 5 mg/mL of the huN901-SPP-DM1 conjugatewas formulated in either (a) PBS, pH 6.5 and stored as a liquid or (b)10 mM sodium succinate, 0.5% sucrose, 0.01% polysorbate 20, 250 mMglycine, pH 5.5, and lyophilized as described in Example 6. Samples wereincubated at 4° C. and 25° C. for 6 months, after which they were testedfor the presence of particles, conjugate dimers, and free drug speciesas described herein. The results of these analyses are set forth inTable 10. TABLE 10 Appearance Particles % Dimer % Free Drug 6 month (>5μm) Time 6 months Time 6 months Formulation Time 0 4° C. 25° C. 25° C.Zero 4° C. 25° C. Zero 4° C. 25° C. 7A clear particulates clear 1122 5.28.5 10.1 1.1 1.6 4.9 liquid and sediments (Comparative) 7B White solidWhite solid White solid 24 3.8 4.1 4.6 0.6 0.6 0.9 lyophilized cake;20-sec cake; 19-sec cake; 15-sec (Invention) reconstitutionreconstitution reconstitution time, clear time, clear time, clearsolution, no solution, no solution, no particulate particulateparticulate

These results demonstrate the stability of the inventive lyophilizedcomposition, as evidenced by the reduction in particles, conjugatedimer, and free drug as compared to the liquid compositions formulatedin PBS.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1. A composition comprising (i) a therapeutically effective amount of aconjugate comprising an antibody specific to a human CD19 antigenchemically coupled to a maytansinoid, (ii) a buffering agent, (iii) asurfactant, (iv) a tonicifying amount of sodium chloride, and (v) water,wherein the composition has a pH of about 5-6.
 2. The composition ofclaim 1, wherein the antibody is a monoclonal antibody.
 3. Thecomposition of claim 2, wherein the antibody is a humanized monoclonalantibody.
 4. The composition of claim 2, wherein the antibody is huB4.5. The composition of claim 1, wherein the maytansinoid comprises athiol group.
 6. The composition of claim 5, wherein the maytansinoid isN²′-deacetyl-N²′-(3-mercapto-1-oxopropyl)-maytansine (DM1).
 7. Thecomposition of claim 5, wherein the maytansinoid isN-deacetyl-N²′-(4-mercapto-4-methyl-1-oxopentyl)-maytansine (DM4). 8.The composition of claim 1, wherein the antibody is chemically coupledto the maytansinoid via chemical bonds selected from the groupconsisting of disulfide bonds, acid labile bonds, photolabile bonds,peptidase labile bonds, thioether bonds, and esterase labile bonds. 9.The composition of claim 1, wherein the concentration of the conjugatein the composition is about 0.1 mg/mL to about 5 mg/mL.
 10. Thecomposition of claim 9, wherein the concentration of the conjugate inthe composition is about 1 mg/mL to about 5 mg/mL.
 11. The compositionof claim 9 wherein the concentration of the conjugate in the compositionis about 5 mg/mL.
 12. The composition of claim 1, wherein the bufferingagent is selected from the group consisting of a citrate buffer, anacetate buffer, a succinate buffer, a phosphate buffer, and a histidinebuffer.
 13. The composition of claim 12, wherein the concentration ofthe buffering agent in the composition is about 2 mM to about 50 mM. 14.The composition of claim 13, wherein the composition comprises a sodiumcitrate buffer at a concentration of about 10 mM and the pH of thecomposition is about 5.5.
 15. The composition of claim 1, wherein thesurfactant is polysorbate 20 or polysorbate
 80. 16. The composition ofclaim 15, wherein the concentration of the surfactant in the compositionis about 0.002% to about 0.1% of the total volume of the composition.17. The composition of claim 16, wherein the surfactant is polysorbate20.
 18. The composition of claim 17, wherein the concentration ofpolysorbate 20 in the composition is about 0.01% of the total volume ofthe composition.
 19. The composition of claim 1, wherein theconcentration of sodium chloride in the composition is about 50 mM toabout 500 mM.
 20. The composition of claim 19, wherein the concentrationof sodium chloride in the composition is about 100 mM to about 200 mM.21. The composition of claim 20, wherein the concentration of sodiumchloride in the composition is about 120 mM.
 22. The composition ofclaim 1, wherein the composition further comprises an antioxidant. 23.The composition of claim 22, wherein the concentration of theantioxidant in the composition is about 100 μM to about 100 mM.
 24. Thecomposition of claim 23, wherein the antioxidant is selected from thegroup consisting of superoxide dismutase, glutathione peroxidase,tocotrienols, polyphenols, zinc, manganese, selenium, vitamin C, vitaminE, beta carotene, cysteine, and methionine.
 25. The composition of claim24, wherein the antioxidant is methionine.
 26. The composition of claim25, wherein the concentration of methionine in the composition is about10 mM.
 27. The composition of claim 1, wherein the composition comprisessucrose.
 28. The composition of claim 27, wherein the concentration ofsucrose in the composition is about 0.1% to about 10% of the totalvolume of the composition.
 29. The composition of claim 28, wherein theconcentration of sucrose in the composition is about 5% of the totalvolume of the composition.
 30. A packaged composition comprising asealed container and dispersed therein the composition of claim 1 and aninert gas overlay.
 31. The packaged composition of claim 30, wherein theinert gas is nitrogen or argon.
 32. A method for killing a cell in ahuman comprising administering to the human a composition comprising (i)a therapeutically effective amount of the conjugate comprising anantibody specific to a human CD 19 antigen chemically coupled to amaytansinoid, (ii) a buffering agent, (iii) a surfactant, (iv) atonicifying amount of sodium chloride, and (v) water, wherein thecomposition has a pH of about 5-6, such that the antibody binds to thesurface of the cell and the cytotoxicity of the maytansinoid isactivated, whereby the cell is killed.
 33. The method of claim 32,wherein the cell is a tumor cell.
 34. The method of claim 32, whereinthe antibody is a monoclonal antibody.
 35. The method of claim 34,wherein the antibody is a humanized monoclonal antibody.
 36. The methodof claim 35, wherein the antibody is huB4.
 37. The method of claim 32,wherein the maytansinoid comprises a thiol group.
 38. The method ofclaim 37, wherein the maytansinoid is DM1.
 39. The method of claim 37,wherein the maytansinoid is DM4.
 40. The method of claim 32, wherein theantibody is chemically coupled to the maytansinoid via chemical bondsselected from the group consisting of disulfide bonds, acid labilebonds, photolabile bonds, peptidase labile bonds, thioether bonds, andesterase labile bonds.
 41. The method of claim 32, wherein the antibodybinds to an antigen present on the surface of the cell.
 42. The methodof claim 32, wherein the concentration of the conjugate in thecomposition is about 0.1 mg/mL to about 5 mg/mL.
 43. The method of claim42, wherein the concentration of the conjugate in the composition isabout 1 mg/mL or more.
 44. The method of claim 42, wherein theconcentration of the conjugate in the composition is about 5 mg/mL. 45.The method of claim 32, wherein the buffering agent is selected from thegroup consisting of a citrate buffer, an acetate buffer, a succinatebuffer, a phosphate buffer, and a histidine buffer.
 46. The method ofclaim 32, wherein the surfactant is polysorbate 20 or polysorbate 80.47. The method of claim 46, wherein the surfactant is polysorbate 20.48. The method of claim 32, wherein the composition further comprises anantioxidant.
 49. The method of claim 48, wherein the antioxidant isselected from the group consisting of superoxide dismutase, glutathioneperoxidase, tocotrienols, polyphenols, zinc, manganese, selenium,vitamin C, vitamin E, beta carotene, cysteine, and methionine.
 50. Themethod of claim 49, wherein the antioxidant is methionine.
 51. Themethod of claim 32, wherein the composition comprises sucrose.
 52. Themethod of claim 32, wherein the composition is administered to the humanintravenously, intraperitoneally, or intratumorally
 53. A lyophilizedcomposition comprising (i) a therapeutically effective amount of aconjugate comprising an antibody specific to a human CD19 antigenchemically coupled to a maytansinoid, (ii) a buffering agent, (iii) asurfactant, (iv) a cryoprotectant, and (v) a bulking agent, wherein thecomposition has a pH of about 5-6 when reconstituted with water.
 54. Thecomposition of claim 53, wherein the antibody is a monoclonal antibody.55. The composition of claim 54, wherein the antibody is a humanizedmonoclonal antibody.
 56. The composition of claim 55, wherein theantibody is huB4.
 57. The composition of claim 53, wherein themaytansinoid comprises a thiol group.
 58. The composition of claim 57,wherein the maytansinoid isN^(2′)-deacetyl-N^(2′)-(3-mercapto-1-oxopropyl)-maytansine (DM1). 59.The composition of claim 57, wherein the maytansinoid isN^(2′)-deacetyl-N^(2′)-(4-mercapto-4-methyl-1-oxopentyl)-maytansine(DM4).
 60. The composition of claim 53, wherein the antibody ischemically coupled to the maytansinoid via chemical bonds selected fromthe group consisting of disulfide bonds, acid labile bonds, photolabilebonds, peptidase labile bonds, thioether bonds, and esterase labilebonds.
 61. The composition of claim 53, wherein the buffering agent isselected from the group consisting of a citrate buffer, an acetatebuffer, a succinate buffer, a phosphate buffer, and a histidine buffer.62. The composition of claim 61, wherein the composition comprises about0.1 mg to about 2 mg of the buffering agent per mg of the conjugate. 63.The composition of claim 62, wherein the composition comprises about 0.3mg sodium succinate buffer per mg of the conjugate.
 64. The compositionof claim 53, wherein the surfactant is polysorbate 20 or polysorbate 80.65. The composition of claim 64, wherein the composition comprises about0.005 mg to about 0.1 mg of the surfactant per mg of the conjugate. 66.The composition of claim 65, wherein the surfactant is polysorbate 20.67. The composition of claim 66, wherein the composition comprises about0.02 mg polysorbate 20 per mg of the conjugate.
 68. The composition ofclaim 53, wherein the composition comprises about 0.5 mg to about 5 mgof the cryoprotectant per mg of the conjugate.
 69. The composition ofclaim 68, wherein the cyroprotectant is sucrose.
 70. The composition ofclaim 69, wherein the composition comprises about 1 mg sucrose per mg ofthe conjugate.
 71. The composition of claim 53, wherein the compositioncomprises about 2 mg to about 20 mg of the bulking agent per mg of theconjugate.
 72. The composition of claim 71, wherein the bulking agent isglycine.
 73. The composition of claim 72, wherein the compositioncomprises about 3.8 mg glycine per mg of the conjugate.
 74. A method forkilling a cell in a human comprising (a) providing the lyophilizedcomposition of claim 53, (b) adding water to the lyophilized compositionto provide a reconstituted composition, and (c) administering thereconstituted composition to the human such that the antibody binds tothe surface of the cell and the cytotoxicity of the maytansinoid isactivated, whereby the cell is killed.
 75. The method of claim 74,wherein the cell is a tumor cell.
 76. The method of claim 74, whereinthe antibody is a monoclonal antibody.
 77. The method of claim 76,wherein the antibody is a humanized monoclonal antibody.
 78. The methodof claim 77, wherein the antibody is huB4.
 79. The method of claim 74,wherein the maytansinoid comprises a thiol group.
 80. The method ofclaim 79, wherein the maytansinoid is DM1.
 81. The method of claim 79,wherein the maytansinoid is DM4.
 82. The method of claim 74, wherein theantibody is chemically coupled to the maytansinoid via chemical bondsselected from the group consisting of disulfide bonds, acid labilebonds, photolabile bonds, peptidase labile bonds, thioether bonds, andesterase labile bonds.
 83. The method of claim 74, wherein the antibodybinds to an antigen present on the surface of the cell
 84. The method ofclaim 74, wherein the concentration of the conjugate in thereconstituted composition is about 0.1 mg/mL to about 5 mg/mL.
 85. Themethod of claim 84, wherein the concentration of the conjugate in thecomposition is about 1 mg/mL or more.
 86. The method of claim 85,wherein the concentration of the conjugate in the reconstitutedcomposition is about 5 mg/mL.
 87. The method of claim 74, wherein thebuffering agent is selected from the group consisting of a citratebuffer, an acetate buffer, a succinate buffer, a phosphate buffer, and ahistidine buffer.
 88. The method of claim 87, wherein the concentrationof the buffering agent in the reconstituted composition is about 2 mM toabout 50 mM.
 89. The method of claim 88, wherein the reconstitutedcomposition comprises a sodium succinate buffer at a concentration ofabout 10 mM and the pH of the reconstituted composition
 90. The methodof claim 74, wherein the surfactant is polysorbate 20 or polysorbate 80.91. The method of claim 90, wherein the concentration of the surfactantin the reconstituted composition is about 0.002% to about 0.1% of thetotal volume of the reconstituted composition.
 92. The method of claim90, wherein the surfactant is polysorbate
 20. 93. The method of claim92, wherein the concentration of polysorbate 20 in the reconstitutedcomposition is about 0.01% of the total volume of the reconstitutedcomposition.
 94. The method of claim 74, wherein the concentration ofthe cryoprotectant in the reconstituted composition is about
 0. 1% toabout 3% of the total volume of the reconstituted composition.
 95. Themethod of claim 94, wherein the cyroprotectant is sucrose.
 96. Themethod of claim 95, wherein the concentration of sucrose in thereconstituted composition is about 0.5% of the total volume of thereconstituted composition.
 97. The method of claim 74, wherein theconcentration of the bulking agent in the reconstituted composition isabout 50 mM to about 500 mM.
 98. The method of claim 97, wherein thebulking agent is glycine.
 99. The method of claim 98, wherein theconcentration of glycine in the reconstituted composition is about 250mM.
 100. The method of claim 74, wherein the composition is administeredto the human intravenously, intraperitoneally, or intratumorally.
 101. Acomposition comprising (i) a therapeutically effective amount of aconjugate comprising an antibody specific to a human CD19 antigenchemically coupled to a maytansinoid, (ii) a buffering agent, (iii) atonicifying amount of sodium chloride, and (iv) water, wherein thecomposition has a pH of about 5-6.
 102. A method for killing a cell in ahuman comprising administering to the human a composition comprising (i)a therapeutically effective amount of a conjugate comprising an antibodyspecific to a human CD19 antigen chemically coupled to a maytansinoid,(ii) a buffering agent, (iii) a tonicifying amount of sodium chloride,and (iv) water, wherein the composition has a pH of about 5-6, such thatthe antibody binds to the surface of the cell and the cytotoxicity ofthe maytansinoid is activated, whereby the cell is killed.
 103. Alyophilized composition comprising (i) a therapeutically effectiveamount of a conjugate comprising an antibody specific to a CD19 antigenchemically coupled to a maytansinoid, (ii) a buffering agent, (iii) acryoprotectant, and (iv) a bulking agent, wherein the composition has apH of about 5-6 when reconstituted with water.
 104. The composition ofclaim 4, wherein the composition comprises (i) about 5 mg/mL of aconjugate comprising huB4 chemically coupled to DM1, (ii) about 10 mMsodium citrate buffer, (iii) about 0.01% polysorbate 20, (iv) about 120mM sodium chloride, and (v) water, wherein the pH of the composition isabout 5.5.